Microglia-Secreted Galectin-3 Acts as a Toll-like Receptor 4 Ligand and Contributes to Microglial Activation

•Gal3 acts as an endogenous TLR4 ligand with a Kd value around 1 μM•Gal3 can initiate a TLR4-dependent inflammatory response in microglia•Gal3 is required for complete activation of TLR4 upon LPS treatment•Gal3-TLR4 interaction is confirmed in vivo and in stroke patients Inflammatory response induced by microglia plays a critical role in the demise of neuronal populations in neuroinflammatory diseases. Although the role of toll-like receptor 4 (TLR4) in microglia’s inflammatory response is fully acknowledged, little is known about endogenous ligands that trigger TLR4 activation. Here, we report that galectin-3 (Gal3) released by microglia acts as an endogenous paracrine TLR4 ligand. Gal3-TLR4 interaction was further confirmed in a murine neuroinflammatory model (intranigral lipopolysaccharide [LPS] injection) and in human stroke subjects. Depletion of Gal3 exerted neuroprotective and anti-inflammatory effects following global brain ischemia and in the neuroinflammatory LPS model. These results suggest that Gal3-dependent-TLR4 activation could contribute to sustained microglia activation, prolonging the inflammatory response in the brain. Inflammatory response induced by microglia plays a critical role in the demise of neuronal populations in neuroinflammatory diseases. Although the role of toll-like receptor 4 (TLR4) in microglia’s inflammatory response is fully acknowledged, little is known about endogenous ligands that trigger TLR4 activation. Here, we report that galectin-3 (Gal3) released by microglia acts as an endogenous paracrine TLR4 ligand. Gal3-TLR4 interaction was further confirmed in a murine neuroinflammatory model (intranigral lipopolysaccharide [LPS] injection) and in human stroke subjects. Depletion of Gal3 exerted neuroprotective and anti-inflammatory effects following global brain ischemia and in the neuroinflammatory LPS model. These results suggest that Gal3-dependent-TLR4 activation could contribute to sustained microglia activation, prolonging the inflammatory response in the brain. The inflammatory response driven by microglia is a key element in brain ischemia (Lambertsen et al., 2012Lambertsen K.L. Biber K. Finsen B. Inflammatory cytokines in experimental and human stroke.J. Cereb. Blood Flow Metab. 2012; 32: 1677-1698Crossref PubMed Scopus (520) Google Scholar) and in neurodegenerative disorders (Burguillos et al., 2011Burguillos M.A. Deierborg T. Kavanagh E. Persson A. Hajji N. Garcia-Quintanilla A. Cano J. Brundin P. Englund E. Venero J.L. Joseph B. Caspase signalling controls microglia activation and neurotoxicity.Nature. 2011; 472: 319-324Crossref PubMed Scopus (449) Google Scholar, Saijo and Glass, 2011Saijo K. Glass C.K. Microglial cell origin and phenotypes in health and disease.Nat. Rev. Immunol. 2011; 11: 775-787Crossref PubMed Scopus (783) Google Scholar). 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Tanaka H. et al.Pharmacological inhibition of TLR4-NOX4 signal protects against neuronal death in transient focal ischemia.Sci Rep. 2012; 2: 896Crossref PubMed Scopus (71) Google Scholar). Despite extensive research, only very few endogenous ligands for TLR4 have been described so far (Chen and Nuñez, 2010Chen G.Y. Nuñez G. Sterile inflammation: sensing and reacting to damage.Nat. Rev. Immunol. 2010; 10: 826-837Crossref PubMed Scopus (2141) Google Scholar). Galectins represent a protein family with at least 15 members that have significant sequence similarity in their carbohydrate-recognition domain (CRD) and bind to β-galactosides with varying affinities and specificities (Barondes et al., 1994Barondes S.H. Castronovo V. Cooper D.N. Cummings R.D. Drickamer K. Feizi T. Gitt M.A. Hirabayashi J. Hughes C. Kasai K. et al.Galectins: a family of animal beta-galactoside-binding lectins.Cell. 1994; 76: 597-598Abstract Full Text PDF PubMed Scopus (1115) Google Scholar, Leffler et al., 2004Leffler H. Carlsson S. Hedlund M. Qian Y. Poirier F. Introduction to galectins.Glycoconj. J. 2004; 19: 433-440Crossref PubMed Scopus (533) Google Scholar). Galectins are classified into three subgroups (1) proto, (2) chimera, and (3) tandem repeat based on their molecular architecture. The proto-type and tandem-repeat-type families comprise proteins with one and two CRDs on a single polypeptide chain, respectively (Kasai and Hirabayashi, 1996Kasai K. Hirabayashi J. Galectins: a family of animal lectins that decipher glycocodes.J. Biochem. 1996; 119: 1-8Crossref PubMed Scopus (458) Google Scholar). Galectin-3 (Gal3) is the only known member of the chimera-type family comprising a C-terminal CRD and N-terminal non-CRD for carbohydrate binding and increased self-association, respectively (Lepur et al., 2012Lepur A. Salomonsson E. Nilsson U.J. Leffler H. Ligand induced galectin-3 protein self-association.J. Biol. Chem. 2012; 287: 21751-21756Crossref PubMed Scopus (112) Google Scholar). Gal3 is known to be involved in the inflammatory response, and its expression is increased in microglial cells upon various neuroinflammatory stimuli as, for instance, the process of ischemic injury (Lalancette-Hébert et al., 2012Lalancette-Hébert M. Swarup V. Beaulieu J.M. Bohacek I. Abdelhamid E. Weng Y.C. Sato S. Kriz J. Galectin-3 is required for resident microglia activation and proliferation in response to ischemic injury.J. Neurosci. 2012; 32: 10383-10395Crossref PubMed Scopus (198) Google Scholar, Satoh et al., 2011aSatoh K. Niwa M. Binh N.H. Nakashima M. Kobayashi K. Takamatsu M. Hara A. Increase of galectin-3 expression in microglia by hyperthermia in delayed neuronal death of hippocampal CA1 following transient forebrain ischemia.Neurosci. Lett. 2011; 504: 199-203Crossref PubMed Scopus (29) Google Scholar, Satoh et al., 2011bSatoh K. Niwa M. Goda W. Binh N.H. Nakashima M. Takamatsu M. Hara A. Galectin-3 expression in delayed neuronal death of hippocampal CA1 following transient forebrain ischemia, and its inhibition by hypothermia.Brain Res. 2011; 1382: 266-274Crossref PubMed Scopus (49) Google Scholar, Wesley et al., 2013Wesley U.V. Vemuganti R. Ayvaci E.R. Dempsey R.J. Galectin-3 enhances angiogenic and migratory potential of microglial cells via modulation of integrin linked kinase signaling.Brain Res. 2013; 1496: 1-9Crossref PubMed Scopus (48) Google Scholar). Gal3 can be found in the cytoplasm, nucleus, and membranes (Shimura et al., 2004Shimura T. Takenaka Y. Tsutsumi S. Hogan V. Kikuchi A. Raz A. Galectin-3, a novel binding partner of beta-catenin.Cancer Res. 2004; 64: 6363-6367Crossref PubMed Scopus (192) Google Scholar) and can be released into the extracellular space upon certain stimuli such as lipopolysaccharide (LPS) (Li et al., 2008Li Y. Komai-Koma M. Gilchrist D.S. Hsu D.K. Liu F.T. Springall T. Xu D. Galectin-3 is a negative regulator of lipopolysaccharide-mediated inflammation.J. Immunol. 2008; 181: 2781-2789Crossref PubMed Scopus (123) Google Scholar) and interferon γ (IFN-γ) (Jeon et al., 2010Jeon S.B. Yoon H.J. Chang C.Y. Koh H.S. Jeon S.H. Park E.J. Galectin-3 exerts cytokine-like regulatory actions through the JAK-STAT pathway.J. Immunol. 2010; 185: 7037-7046Crossref PubMed Scopus (138) Google Scholar). The different subcellular localizations of Gal3 together with its possible posttranslational modifications are likely to affect the function of Gal3 and explain why rather contradictory effects have been reported, e.g., pro- versus anti-apoptotic (Nakahara et al., 2005Nakahara S. Oka N. Raz A. On the role of galectin-3 in cancer apoptosis.Apoptosis. 2005; 10: 267-275Crossref PubMed Scopus (257) Google Scholar) and pro- versus anti-inflammatory (Jeon et al., 2010Jeon S.B. Yoon H.J. Chang C.Y. Koh H.S. Jeon S.H. Park E.J. Galectin-3 exerts cytokine-like regulatory actions through the JAK-STAT pathway.J. Immunol. 2010; 185: 7037-7046Crossref PubMed Scopus (138) Google Scholar, MacKinnon et al., 2008MacKinnon A.C. Farnworth S.L. Hodkinson P.S. Henderson N.C. Atkinson K.M. Leffler H. Nilsson U.J. Haslett C. Forbes S.J. Sethi T. Regulation of alternative macrophage activation by galectin-3.J. Immunol. 2008; 180: 2650-2658Crossref PubMed Scopus (408) Google Scholar). As an example of this duality of function, it has been reported that Gal3 deficiency aggravates the neuronal damage in the adult mouse brain following transient focal brain ischemia, due to a reduced signaling of insulin-like growth factor receptor in microglia (Lalancette-Hébert et al., 2012Lalancette-Hébert M. Swarup V. Beaulieu J.M. Bohacek I. Abdelhamid E. Weng Y.C. Sato S. Kriz J. Galectin-3 is required for resident microglia activation and proliferation in response to ischemic injury.J. Neurosci. 2012; 32: 10383-10395Crossref PubMed Scopus (198) Google Scholar), whereas in a transgenic mouse model of amyotrophic lateral sclerosis (ALS), the lack of Gal3 increases the inflammatory response (Lerman et al., 2012Lerman B.J. Hoffman E.P. Sutherland M.L. Bouri K. Hsu D.K. Liu F.T. Rothstein J.D. Knoblach S.M. Deletion of galectin-3 exacerbates microglial activation and accelerates disease progression and demise in a SOD1(G93A) mouse model of amyotrophic lateral sclerosis.Brain Behav. 2012; 2: 563-575Crossref PubMed Scopus (65) Google Scholar). In contrast, in a model of global brain ischemia, microglial Gal3 was suggested to contribute to neuronal death in the CA1 subregion of the hippocampus (Satoh et al., 2011aSatoh K. Niwa M. Binh N.H. Nakashima M. Kobayashi K. Takamatsu M. Hara A. Increase of galectin-3 expression in microglia by hyperthermia in delayed neuronal death of hippocampal CA1 following transient forebrain ischemia.Neurosci. Lett. 2011; 504: 199-203Crossref PubMed Scopus (29) Google Scholar, Satoh et al., 2011bSatoh K. Niwa M. Goda W. Binh N.H. Nakashima M. Takamatsu M. Hara A. Galectin-3 expression in delayed neuronal death of hippocampal CA1 following transient forebrain ischemia, and its inhibition by hypothermia.Brain Res. 2011; 1382: 266-274Crossref PubMed Scopus (49) Google Scholar) as well as contribute to the inflammation and severity in experimental autoimmune encephalitis (Jiang et al., 2009Jiang H.R. Al Rasebi Z. Mensah-Brown E. Shahin A. Xu D. Goodyear C.S. Fukada S.Y. Liu F.T. Liew F.Y. Lukic M.L. Galectin-3 deficiency reduces the severity of experimental autoimmune encephalomyelitis.J. Immunol. 2009; 182: 1167-1173Crossref PubMed Scopus (48) Google Scholar). Previous studies have focused on the relationship between Gal3 and members of the TLR family such as TLR2. For example, in differentiated macrophages, Gal3 can form a complex with TLR2 and thereby improves the inflammatory response against C. Albicans (Jouault et al., 2006Jouault T. El Abed-El Behi M. Martínez-Esparza M. Breuilh L. Trinel P.A. Chamaillard M. Trottein F. Poulain D. Specific recognition of Candida albicans by macrophages requires galectin-3 to discriminate Saccharomyces cerevisiae and needs association with TLR2 for signaling.J. Immunol. 2006; 177: 4679-4687Crossref PubMed Scopus (196) Google Scholar). In addition, it has been suggested that Gal3 can act as co-receptor, presenting the Toxoplasma gondii glycosylphosphatidylinositols (GPIs) to TLR2 and TLR4 on macrophages (Debierre-Grockiego et al., 2010Debierre-Grockiego F. Niehus S. Coddeville B. Elass E. Poirier F. Weingart R. Schmidt R.R. Mazurier J. Guérardel Y. Schwarz R.T. Binding of Toxoplasma gondii glycosylphosphatidylinositols to galectin-3 is required for their recognition by macrophages.J. Biol. Chem. 2010; 285: 32744-32750Crossref PubMed Scopus (42) Google Scholar). Furthermore, an interaction between Gal3 and LPS, a known TLR4 ligand, has been reported as well (Li et al., 2008Li Y. Komai-Koma M. Gilchrist D.S. Hsu D.K. Liu F.T. Springall T. Xu D. Galectin-3 is a negative regulator of lipopolysaccharide-mediated inflammation.J. Immunol. 2008; 181: 2781-2789Crossref PubMed Scopus (123) Google Scholar, Mey et al., 1996Mey A. Leffler H. Hmama Z. Normier G. Revillard J.P. The animal lectin galectin-3 interacts with bacterial lipopolysaccharides via two independent sites.J. Immunol. 1996; 156: 1572-1577PubMed Google Scholar). Gal3 and TLR4 are both considered to be independent actors in the initiation and progression of the inflammatory response after brain ischemia. In this study, we demonstrate that Gal3 can act as an endogenous ligand for TLR4. We show that Gal3 can induce, per se, a TLR4-dependent inflammatory response as well as contribute to the full activation of this receptor upon binding to other proinflammatory stimuli, such as LPS. We first set out to determine the effect of Gal3 on the TLR-mediated signaling pathways. To achieve this, we took advantage of an array that monitors the expression of 84 genes involved in the TLRs intracellular signaling pathways. BV2 microglia cells were exposed to endotoxin-free (as confirmed by Limulus amebocyte lysate assay) soluble Gal3 (referred henceforth as sGal3) for 6 hr. In addition, because Gal3 can be rapidly internalized by cells and thereby activate intracellular signaling pathways, we used a so-called “immobilized form” of Gal3 (referred to as iGal3) that only can interact with proteins on the cell surface (e.g., receptors). Due to Gal3’s high hydrophobicity of its N terminus part, it can bind to plastic, allowing the exposure of both domains: its CRD and also its N-terminal site (Sörme et al., 2002Sörme P. Qian Y. Nyholm P.G. Leffler H. Nilsson U.J. Low micromolar inhibitors of galectin-3 based on 3′-derivatization of N-acetyllactosamine.ChemBioChem. 2002; 3: 183-189Crossref PubMed Scopus (97) Google Scholar). Cell culture wells were coated overnight at 4°C with 100 μg/ml of Gal3 and washed three times with PBS to remove unbound Gal3. BV2 microglial cells were then seeded in these Gal3-coated plastic wells for 6 hr before harvesting them. Cells seeded on non-coated wells for 6 hr were used as a negative control. LPS (1 μg/ml) added to the cell culture medium for 6 hr was used as a positive control for TLR4 activation. Thus, BV2 microglia cells were treated with sGal3, iGal3, or LPS and gene expression of the TLRs-signaling pathway investigated. As shown in Figure 1, sGal3 or iGal3 treatment results in statistically significant changes in gene expression as compared to untreated cells. Both induction and repression in gene expression can be observed after either of these treatments. Remarkably, there was significant overlap in microglial gene expression related to TLR4 signaling in responses to either Gal3 or LPS (Figures S1A and S1B). Next, we explored the possibility of a direct physical interaction between Gal3 and TLR4. Using confocal microscopy, Gal3 and TLR4 were found to be colocalized in BV2 cells 1 hr after adding sGal3 (Figure 2A). Under these conditions, TLR4 was immunoprecipitated and Gal3 was found to be part of the resulting immune complexes (Figure 2B). Gal3 interaction with glycoproteins is complex, and the initial binding of the CRD often triggers a subsequent self-association of Gal3, sometimes resulting in crosslinking and precipitation (Lepur et al., 2012Lepur A. Salomonsson E. Nilsson U.J. Leffler H. Ligand induced galectin-3 protein self-association.J. Biol. Chem. 2012; 287: 21751-21756Crossref PubMed Scopus (112) Google Scholar). This self-association also involves the canonical carbohydrate recognition site in the CRD but also the N-terminal non-CRD domain of Gal3, which makes it much more efficient, and is also required for most biological effects of Gal3. The apparent affinity of the interaction between TLR4 and Gal3 was determined using microscale thermophoresis (MST). In MST, the thermophoretic mobility of a fluorescently labeled molecule in an infrared-laser-induced microscopic temperature gradient is recorded, yielding a fluorescence time trace from which a normalized fluorescence value (Fnorm) is derived. Changes in the thermophoretic mobility of the molecule upon ligand binding manifest as shifts in the Fnorm values and are used to quantify the affinity of the interactions (Seidel et al., 2013Seidel S.A. Dijkman P.M. Lea W.A. van den Bogaart G. Jerabek-Willemsen M. Lazic A. Joseph J.S. Srinivasan P. Baaske P. Simeonov A. et al.Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.Methods. 2013; 59: 301-315Crossref PubMed Scopus (435) Google Scholar). Accordingly, binding of Gal3 to fluorophore-tagged TLR4 (at a constant concentration of about 120 nM) produced a clear shift in the recorded fluorescence time traces (Figure S2B) with increased Fnorm values for the Gal3-TLR4 complex. The minimal and maximal Fnorm values for the unbound and fully bound state of TLR4, respectively, were used to calculate the fraction of TLR4 bound at each Gal3 concentration. The resulting saturation binding curve (Figure 2C) shows that 50% of TLR4 is bound at about 1.5 μM Gal3. The presence of lactose, a competitive inhibitor of both Gal3 carbohydrate binding and self-association, completely abolished the interaction (purple data points in Figure 2C). Further evidence for the involvement of the Gal3 canonical carbohydrate-binding site was the fact that a mutant, Gal3 R186S, showed interaction with TLR4 at a much-higher concentration with 50% bound at about 45 μM. This mutant reduces affinity of Gal3 for many glycoproteins and for the disaccharide LacNAc, which is the most common minimal galectin-binding moiety in glycoproteins (Lepur et al., 2012Lepur A. Salomonsson E. Nilsson U.J. Leffler H. Ligand induced galectin-3 protein self-association.J. Biol. Chem. 2012; 287: 21751-21756Crossref PubMed Scopus (112) Google Scholar, Salomonsson et al., 2010aSalomonsson E. Carlsson M.C. Osla V. Hendus-Altenburger R. Kahl-Knutson B. Oberg C.T. Sundin A. Nilsson R. Nordberg-Karlsson E. Nilsson U.J. et al.Mutational tuning of galectin-3 specificity and biological function.J. Biol. Chem. 2010; 285: 35079-35091Crossref PubMed Scopus (91) Google Scholar). The Gal3 CRD, lacking the N-terminal domain, also bound TLR4 at about equal concentrations as intact Gal3 (red curve in Figure 2C). To gain further evidence for Gal3-TLR4 interaction, we used fluorescence anisotropy as a separate independent technique. In this technique, the interaction of Gal3 with a fluorescein-tagged saccharide probe is inhibited by increasing concentrations of TLR4 and quantitatively analyzed, as has been done for many other inhibitors before (Lepur et al., 2012Lepur A. Salomonsson E. Nilsson U.J. Leffler H. Ligand induced galectin-3 protein self-association.J. Biol. Chem. 2012; 287: 21751-21756Crossref PubMed Scopus (112) Google Scholar, Salomonsson et al., 2010bSalomonsson E. Larumbe A. Tejler J. Tullberg E. Rydberg H. Sundin A. Khabut A. Frejd T. Lobsanov Y.D. Rini J.M. et al.Monovalent interactions of galectin-1.Biochemistry. 2010; 49: 9518-9532Crossref PubMed Scopus (51) Google Scholar). The data are presented in the form of percent Gal3 bound to TLR4 to make them more easily comparable to the previous experiment (Figure 2D). This again demonstrated that TLR4 binds both Gal3 and Gal3 CRD, with 50% of Gal3 bound by about 1 μM TLR4, and also shows that TLR4 competes for the canonical carbohydrate-binding site of Gal3. The data also provided insight into TLR4-induced self-association of Gal3. The slope of the binding curve in Figure 2C, where fixed TLR4 is titrated with a range of Gal3 concentrations, was consistent with a Hill coefficient of above 2 for intact Gal3 but was about 1 for the CRD. In Figure 2D, where fixed Gal3 is titrated with a range of TLR4 concentrations, the Hill coefficient for intact Gal3 was about 0.4, whereas for the CRD, it was again about 1 (Table S1). This indicates that intact Gal3 binds with apparent positive cooperativity and/or in an event with stoichiometry of greater than two Gal3 per TLR4, whereas the CRD binds in simple 1:1 interactions to one or more independent sites on TLR4. Addition of Gal3 concentration >∼1 μM to fluorescent TLR4 at 120 nM caused precipitation, as measured by removal of fluorescence by centrifugation of the samples before loading into capillaries that are used for the MST measurements (Figure S2A). This observation probably also explains the gradual fluorescence increase in un-centrifuged samples (Figure S2A) and the wavy line shapes of the fluorescence time traces recorded in the MST experiment (Figure S2B). However, the aggregation did not prevent obtaining highly reproducible binding curves that could be used for quantitative analysis of the interaction (Figure 2C). The different methods, hence, demonstrate that Gal3 interacts directly with TLR4 at physiologically relevant concentrations and also at the Gal3 concentrations (1 μM) used in the cell experiment here. All galectin family members have in common a canonical CRD with high-sequence homology. Galectin-1 and galectin-4 were chosen as examples of the proto and tandem repeat families, respectively, and they also bind to TLR4 in MST experiments but with lower apparent affinities of about 8 and 14 μM, respectively, and Hill coefficients of about 1, indicating a lower cooperativity (Figure S2F). Contradictory reports suggest that Gal3 can play both proinflammatory and anti-inflammatory roles. Gal3 has been shown to elicit a proinflammatory (M1) response per se (Jeon et al., 2010Jeon S.B. Yoon H.J. Chang C.Y. Koh H.S. Jeon S.H. Park E.J. Galectin-3 exerts cytokine-like regulatory actions through the JAK-STAT pathway.J. Immunol. 2010; 185: 7037-7046Crossref PubMed Scopus (138) Google Scholar) or amplify a pre-existent proinflammatory reaction (Devillers et al., 2013Devillers A. Courjol F. Fradin C. Coste A. Poulain D. Pipy B. Bernardes E.S. Jouault T. Deficient beta-mannosylation of Candida albicans phospholipomannan affects the proinflammatory response in macrophages.PLoS ONE. 2013; 8: e84771Crossref PubMed Scopus (16) Google Scholar) in macrophages. Similarly, we have recently demonstrated that Gal3 is involved in the proinflammatory response triggered by α-synuclein in microglial cells (Boza-Serrano et al., 2014Boza-Serrano A. Reyes J.F. Rey N.L. Leffler H. Bousset L. Nilsson U. Brundin P. Venero J. Burguillos M. Deierborg T. The role of Galectin-3 in α-synuclein-induced microglial activation.Acta Neuropathol Commun. 2014; 2: 156PubMed Google Scholar). Other studies have, however, suggested that Gal3 is involved in the alternative activation of macrophages and microglia (Hoyos et al., 2014Hoyos H.C. Rinaldi M. Mendez-Huergo S.P. Marder M. Rabinovich G.A. Pasquini J.M. Pasquini L.A. Galectin-3 controls the response of microglial cells to limit cuprizone-induced demyelination.Neurobiol. Dis. 2014; 62: 441-455Crossref PubMed Scopus (61) Google Scholar, MacKinnon et al., 2008MacKinnon A.C. Farnworth S.L. Hodkinson P.S. Henderson N.C. Atkinson K.M. Leffler H. Nilsson U.J. Haslett C. Forbes S.J. Sethi T. Regulation of alternative macrophage activation by galectin-3.J. Immunol. 2008; 180: 2650-2658Crossref PubMed Scopus (408) Google Scholar). In order to clarify the effect of Gal3 per se on microglial cells, BV2 cells were treated with sGal3 and several phenotypical markers were analyzed, including the expression of inducible nitric oxide synthase (iNOS) (M1 phenotype), CD206, TGF-β, Ym 1/2, arginase-1 activity (M2 phenotype), and CD45 (phosphatase that can inhibit the proinflammatory response; Starossom et al., 2012Starossom S.C. Mascanfroni I.D. Imitola J. Cao L. Raddassi K. Hernandez S.F. Bassil R. Croci D.O. Cerliani J.P. Delacour D. et al.Galectin-1 deactivates classically activated microglia and protects from inflammation-induced neurodegeneration.Immunity. 2012; 37: 249-263Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar). We observed that sGal3 treatment induced iNOS expression (Figures 3B and 3C ) and an overall trend to decrease the different M2 markers, although only arginase activity and CD206 expression reached statistical significance (Figures S3A and S3B). These data support the view that Gal3 stimulates a proinflammatory M1 phenotype in microglia. The similarities between the changes in gene expression induced by Gal3 and LPS, which acts as a TLR4 ligand (Figure 1), and the physical interaction between Gal3 and TLR4 made us think that Gal3 could be inducing a TLR4-dependent inflammatory response. To explore this possibility, the expression of TLR4 was silenced in BV2 microglial cells using small interfering RNA (siRNA) (Figure 3A). Interestingly, silencing of TLR4 in BV2 cells leads to a reduction in the iNOS protein expression upon LPS, sGal3, and iGal3 treatments (Figures 3B–3E), suggesting that these stimuli share a common TLR4-dependent signaling pathway. The silencing of MyD88, a downstream protein triggered by activation of TLR4, shows as well a decrease in iNOS expression upon sGal3 treatment in BV2 cells (Figures S3C and S3D). To validate the TLR4 dependency of the Gal3 response, the release of several cytokines (i.e., TNF-α and interleukins [IL-1β, IL-4, IL-5, IL-10, and IL-12]) were investigated in primary microglia cultures derived from wild-type and TLR4 knockout mice upon sGal3 and iGal3 treatment. The release of the above-mentioned cytokines was found to be increased upon both types of Gal3 treatment in wild-type microglia (Figures 3F–3K). In contrast, the increases in cytokines released upon Gal3 treatments were abrogated in primary microglial cells originating from TLR4 knockout mice (Figures 3F–3K), demonstrating that TLR4 is essential for Gal3-induced cytokine release. In the case of IL-10 and TNF-α, we observed that their decrease is not complete in TLR4 knockout mice, which suggests also that Gal3 may be interacting also with other receptors other than TLR4 such as for example TLR2 (Jouault et al., 2006Jouault T. El Abed-El Behi M. Martínez-Esparza M. Breuilh L. Trinel P.A. Chamaillard M. Trottein F. Poulain D. Specific recognition of Candida albicans by macrophages requires galectin-3 to discriminate Saccharomyces cerevisiae and needs association with TLR2 for signaling.J. Immunol. 2006; 177: 4679-4687Crossref PubMed Scopus (196) Google Scholar). We recently uncovered that the orderly activation of caspase-8 and caspase-3/7 contributes to the activation of microglia by several proinflammatory stimuli including LPS (Burguillos et al., 2011Burguillos M.A. Deierborg T. Kavanagh E. Persson A. Hajji N. Garcia-Quintanilla A. Cano J. Brundin P. Englund E. Venero J.L. Joseph B. Caspase signalling controls microglia activation and neurotoxicity.Nature. 2011; 472: 319-324Crossref PubMed Scopus (449) Google Scholar, Venero et al., 2011Venero J.L. Burguillos M.A. Brundin P. Joseph B. The executioners sing a new song: killer caspases activate microglia.Cell Death Differ. 2011; 18: 1679-1691Crossref PubMed Scopus (45) Google Scholar). Because both Gal3 and LPS can act as TLR4 ligands, we next examined whether Gal3 induces the activation of these caspases. Indeed, both sGal3 (Figure 4A) and iGal3 treatments (Figure 4B) induced DEVDase activity (caspase-3/7 activation) and IETDase activity (caspase-8 activation) as early as 6 hr after sGal3 and 1 hr after iGal3 treatment. In accordance with the TLR4 dependency of Gal3 response, silencing of TLR4 expression using siRNA abrogated the increase of both caspase-3/7 and casapase-8 activities after either sGal3 (Figure 4C) or iGal3 (Figure 4D) treatment. We previously demonstrated that the TLR4-dependent activation of these caspases during microglia activation did not lead to cell death (Burguillos et al., 2011Burguillos M.A. Deierborg T. Kavanagh E. Persson A. Hajji N. Garcia-Quintanilla A. Cano J. Brundin P. Englund E. Venero J.L. Joseph B. Caspase signalling controls microglia activation and neurotoxicity.Nature. 2011; 472: 319-324Crossref PubMed Scopus (449) Google Scholar). We confirm here the absence of apoptotic cell death upon Gal3 treatment using a panel of methods (Figures S4A–S4D). Some reports indicated that Gal3 can affect the cell cycle (Lin et al., 2002Lin H.M. Pestell R.G. Raz A. Kim H.R. Galectin-3 enhances cyclin D(1) promoter activity through SP1 and a cAMP-responsive element in human breast epithelial cells.Oncogene. 2002; 21: 8001-8010Crossref PubMed Scopus (128) Google Scholar). However, we di